Polypeptides Having Xylanase Activity And Polynucleotides Thereof

ABSTRACT

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/668,599, filed Mar. 25, 2015, which is a divisional applicationof U.S. application Ser. No. 13/151,128, filed Jun. 1, 2011, now U.S.Pat. No. 8,999,694, which is a divisional application of U.S.application Ser. No. 11/054,191, filed Feb. 9, 2005, now U.S. Pat. No.7,960,160, which claims the benefit of U.S. Provisional Application No.60/544,461, filed Feb. 12, 2004, U.S. Provisional Application No.60/544,429, filed Feb. 12, 2004, and U.S. Provisional Application No.60/544,431, filed Feb. 12, 2004, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to isolated polypeptides having xylanaseactivity and isolated polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods for producingand using the polypeptides.

Description of the Related Art

Xylan, a major component of plant hemicellulose, is a polymer ofD-xylose linked by beta-1,4-xylosidic bonds. Xylan can be degraded toxylose and xylo-oligomers by acid or enzymatic hydrolysis. Enzymatichydrolysis of xylan produces free sugars without the by-products formedwith acid (e.g., furans).

Enzymes capable of degrading xylan and other plant cell wallpolysaccharides are important for the food industry, primarily forbaking and in fruit and vegetable processing such as fruit juiceproduction or wine making, where their ability to catalyse thedegradation of the backbone or side chains of the plant cell wallpolysaccharide is utilized (Visser et al., Xylans and Xylanases,Proceedings of an International Symposium, Wageningen, The Netherlands,Elsevier Science Publishers, 1992).

Other applications for xylanases are enzymatic breakdown of agriculturalwastes for production of alcohol fuels, enzymatic treatment of animalfeeds for hydrolysis of pentosans, manufacturing of dissolving pulpsyielding cellulose, and bio-bleaching of wood pulp [Detroym R. W. In:Organic Chemicals from Biomass, (CRC Press, Boca Raton, Fla., 1981)19-41; Paice and Jurasek, J. Wood Chem. Technol. 4: 187-198; Pommier andFuentes, 1989, Tappi Journal 187-191; Senior et al., 1988, Biotechnol.Letters 10: 907-9121].

WO 92/17573 discloses a substantially pure xylanase derived fromHumicola insolens and recombinant DNA encoding said xylanase for as abaking agent, a feed additive, and in the preparation of paper and pulp.

WO 92/01793 discloses a xylanase derived from Aspergillus tubigensis. Itis mentioned, but not shown that related xylanases may be derived fromother filamentous fungi, examples of which are Aspergillus,Disporotrichum, Penicillium, Neurospora, Fusarium and Trichoderma. Thexylanases are stated to be useful in the preparation of bread or animalfeed, in brewing and in reducing viscosity or improving filterability ofcereal starch.

Shei et al., 1985, Biotech. and Bioeng. Vol. XXVII, pp. 533-538, andFournier et al., 1985, Biotech. and Bioeng. Vol. XXVII, pp. 539-546,describe purification and characterization of endoxylanases isolatedfrom Aspergillus niger.

WO 91/19782 and EP 463 706 discloses xylanase derived from Aspergillusniger origin and the recombinant production thereof for use in baking,brewing, paper-making, and treatment of agricultural waste.

WO 03/012071 discloses nucleotide sequences of Aspergillus fumigatusxylanases.

It is an object of the present invention to provide new polypeptideshaving xylanase activity and nucleic acids encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having xylanaseactivity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 65%identity with amino acids 18 to 364 of SEQ ID NO: 2, at least 85%identity with amino acids 20 to 323 of SEQ ID NO: 4, or at least 80%identity with amino acids 20 to 397 of SEQ ID NO: 6;

(b) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under at least low stringency conditions with (i) nucleotides52 to 1145 of SEQ ID NO: 1, (ii) the cDNA sequence contained innucleotides 52 to 1145 of SEQ ID NO: 1, or (iii) a complementary strandof (i) or (ii); or under at least medium-high stringency conditions with(iv) nucleotides 58 to 1400 of SEQ ID NO: 3 or nucleotides 107 to 1415of SEQ ID NO: 5, (v) the cDNA sequence contained in nucleotides 58 to1400 of SEQ ID NO: 3 or nucleotides 107 to 1415 of SEQ ID NO: 5, or (vi)a complementary strand of (iv) or (v); and

(c) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of amino acids 18 to 364 of SEQ IDNO: 2, amino acids 20 to 323 of SEQ ID NO: 4, or amino acids 20 to 397of SEQ ID NO: 6.

The present invention also relates to isolated polynucleotides encodingpolypeptides having xylanase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide having an amino acidsequence which has at least 65% identity with amino acids 18 to 364 ofSEQ ID NO: 2, at least 85% identity with amino acids 20 to 323 of SEQ IDNO: 4, or at least 80% identity with amino acids 20 to 397 of SEQ ID NO:6;

(b) a polynucleotide having at least 65% identity with nucleotides 52 to1145 of SEQ ID NO: 1, at least 85% identity with nucleotides 58 to 1400of SEQ ID NO: 3, or at least 80% identity with nucleotides 107 to 1415of SEQ ID NO: 5; and

(c) a polynucleotide which hybridizes under at least low stringencyconditions with (i) nucleotides 52 to 1145 of SEQ ID NO: 1, (ii) thecDNA sequence contained in nucleotides 52 to 1145 of SEQ ID NO: 1, or(iii) a complementary strand of (i) or (ii); or under at leastmedium-high stringency conditions with (iv) nucleotides 58 to 1400 ofSEQ ID NO: 3 or nucleotides 107 to 1415 of SEQ ID NO: 5, (v) the cDNAsequence contained in nucleotides 58 to 1400 of SEQ ID NO: 3 ornucleotides 107 to 1415 of SEQ ID NO: 5, or (vi) a complementary strandof (iv) or (v).

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides.

The present invention also relates to methods for producing such apolypeptide having xylanase activity comprising: (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of using the polypeptidesin treating pulp, in processes for producing xylose orxylo-oligosaccharide, as feed enhancing enzymes that improve feeddigestibility, in baking, and in brewing.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide consisting ofnucleotides 1 to 51 of SEQ ID NO: 1, nucleotides 1 to 57 of SEQ ID NO:3, or nucleotides 1 to 106 of SEQ ID NO: 5 or the cDNA thereof, whereinthe gene is foreign to the nucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus fumigatus Family GH10A xylanase (SEQ ID NOs:1 and 2, respectively). The predicted signal peptide is underlined andpredicted introns are italicized. FIG. 1B is a continuation of thesequences shown in FIG. 1A.

FIG. 2 shows a restriction map of pAlLo1.

FIG. 3 shows a restriction map of pBM121b.

FIG. 4 shows a restriction map of pBM120a.

FIG. 5 shows a restriction map of pSMO208.

FIG. 6 shows a restriction map of pSMO210.

FIGS. 7A and 7B show the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus fumigatus Family GH10B xylanase (SEQ ID NOs:3 and 4, respectively). The predicted signal peptide is underlined andpredicted introns are italicized. FIG. 7B is a continuation of thesequences shown in FIG. 7A.

FIG. 8 shows a restriction map of pJLin162.

FIGS. 9A and 9B show the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus fumigatus Family GH10C xylanase (SEQ ID NOs:5 and 6, respectively). The predicted signal peptide is underlined andpredicted introns are italicized. FIG. 9B is a continuation of thesequences shown in FIG. 9A.

FIG. 10 shows a restriction map of pHyGe009.

FIG. 11 shows a restriction map of pHyGe001.

DEFINITIONS

Xylanase activity: The term “xylanase” is defined herein as a1,4-beta-D-xylan-xylanohydrolase (E.C. 3.2.1.8) which catalyzes theendohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For purposesof the present invention, xylanase activity is determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate buffer pH 6 at 37° C. One unit of xylanase activity is definedas 1.0 μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the xylanase activity of thepolypeptide consisting of the amino acid sequence shown as amino acids18 to 364 of SEQ ID NO: 2, amino acids 20 to 323 of SEQ ID NO: 4, oramino acids 20 to 397 of SEQ ID NO: 6.

Family GH10 xylanase: The term “Family 10 glycoside hydrolase” or“Family GH10” is defined herein as a polypeptide falling into theglycoside hydrolase Family 10 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively associated. It is, therefore,preferred that the substantially pure polypeptide is at least 92% pure,preferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 96% pure, morepreferably at least 97% pure, more preferably at least 98% pure, evenmore preferably at least 99%, most preferably at least 99.5% pure, andeven most preferably 100% pure by weight of the total polypeptidematerial present in the preparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE® MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10 and gap length penaltyof 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE® MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of SEQ ID NO: 2, 4, or 6, or a homologoussequence thereof, wherein the fragment has xylanase activity.

Preferably, a fragment of SEQ ID NO: 2 contains at least 300 amino acidresidues, more preferably at least 315 amino acid residues, and mostpreferably at least 330 amino acid residues.

Preferably, a fragment of SEQ ID NO: 4 contains at least 255 amino acidresidues, more preferably at least 270 amino acid residues, and mostpreferably at least 285 amino acid residues.

Preferably, a fragment of SEQ ID NO: 6 contains at least 320 amino acidresidues, more preferably at least 340 amino acid residues, and mostpreferably at least 360 amino acid residues.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of SEQ ID NO: 1, 3, or 5, or a homologous sequence thereof, whereinthe subsequence encodes a polypeptide fragment having xylanase activity.

Preferably, a subsequence of SEQ ID NO: 1 contains at least 900nucleotides, more preferably at least 945 nucleotides, and mostpreferably at least 990 nucleotides.

Preferably, a subsequence of SEQ ID NO: 3 contains at least 765nucleotides, more preferably at least 810 nucleotides, and mostpreferably at least 855 nucleotides.

Preferably, a subsequence of SEQ ID NO: 5 contains at least 960nucleotides, more preferably at least 1020 nucleotides, and mostpreferably at least 1080 nucleotides.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 20% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG and TGA. The coding sequence may be aDNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of amino acids 18 to 364 ofSEQ ID NO: 2, amino acids 20 to 323 of SEQ ID NO: 4, or amino acids 20to 397 of SEQ ID NO: 6, or a homologous sequence thereof, as well asgenetic manipulation of the DNA encoding that polypeptide. Themodification can be substitutions, deletions and/or insertions of one ormore amino acids as well as replacements of one or more amino acid sidechains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having xylanase activity produced by an organismexpressing a modified nucleotide sequence of SEQ ID NO: 1, 3, or 5, or ahomologous sequence thereof, or the mature coding region thereof. Themodified nucleotide sequence is obtained through human intervention bymodification of the nucleotide sequence disclosed in SEQ ID NO: 1, 3, or5, or a homologous sequence thereof, or the mature coding regionthereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having XylanaseActivity

In a first aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 18 to 364 of SEQ ID NO: 2 (i.e., the maturepolypeptide) of at least 65%, preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 97%, 98%, or 99%, which have xylanaseactivity; a degree of identity to amino acids 20 to 323 of SEQ ID NO: 4(i.e., the mature polypeptide) of at least 85%, preferably at least 90%,more preferably at least 95%, and most preferably at least 97%, 98%, or99%, which have xylanase activity; or a degree of identity to aminoacids 20 to 397 of SEQ ID NO: 6 (i.e., the mature polypeptide) of atleast 80%, preferably at least 85%, more preferably at least 90%, morepreferably at least 95%, and most preferably at least 97%, 98%, or 99%,which have xylanase activity (hereinafter “homologous polypeptides”). Ina preferred aspect, the homologous polypeptides have an amino acidsequence which differs by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from amino acids 18 to 364 of SEQ ID NO: 2,amino acids 20 to 323 of SEQ ID NO: 4, or amino acids 20 to 397 of SEQID NO: 6.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has xylanase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide comprises amino acids 18 to 364of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another preferred aspect, a polypeptidecomprises amino acids 18 to 364 of SEQ ID NO: 2. In another preferredaspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, a polypeptide consists of theamino acid sequence of SEQ ID NO: 2. In another preferred aspect, apolypeptide consists of amino acids 18 to 364 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, a polypeptide consists of aminoacids 18 to 364 of SEQ ID NO: 2.

A polypeptide of the present invention also preferably comprises theamino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or afragment thereof that has xylanase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, a polypeptide comprises amino acids 20 to 323of SEQ ID NO: 4, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another preferred aspect, a polypeptidecomprises amino acids 20 to 323 of SEQ ID NO: 4. In another preferredaspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:4 or an allelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, a polypeptide consists of theamino acid sequence of SEQ ID NO: 4. In another preferred aspect, apolypeptide consists of amino acids 20 to 323 of SEQ ID NO: 4 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, a polypeptide consists of aminoacids 20 to 323 of SEQ ID NO: 4.

A polypeptide of the present invention also preferably comprises theamino acid sequence of SEQ ID NO: 6 or an allelic variant thereof; or afragment thereof that has xylanase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, a polypeptide comprises amino acids 20 to 397of SEQ ID NO: 6, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another preferred aspect, a polypeptidecomprises amino acids 20 to 397 of SEQ ID NO: 6. In another preferredaspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:6 or an allelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, a polypeptide consists of theamino acid sequence of SEQ ID NO: 6. In another preferred aspect, apolypeptide consists of amino acids 20 to 397 of SEQ ID NO: 6 or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another preferred aspect, a polypeptide consists of aminoacids 20 to 397 of SEQ ID NO: 6.

In a second aspect, the present invention relates to isolatedpolypeptides having xylanase activity which are encoded bypolynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) nucleotides 52 to 1145 of SEQ ID NO: 1,nucleotides 58 to 1400 of SEQ ID NO: 3, or nucleotides 107 to 1415 ofSEQ ID NO: 5, (ii) the cDNA sequence contained in nucleotides 52 to 1145of SEQ ID NO: 1, nucleotides 58 to 1400 of SEQ ID NO: 3, or nucleotides107 to 1415 of SEQ ID NO: 5, (iii) a subsequence of (i) or (ii), or (iv)a complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.). A subsequence of SEQ ID NO: 1, 3,or 5 contains at least 100 contiguous nucleotides or preferably at least200 contiguous nucleotides. Moreover, the subsequence may encode apolypeptide fragment which has xylanase activity.

The nucleotide sequence of SEQ ID NO: 1, 3, or 5, or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO: 2, 4, or 6, ora fragment thereof, may be used to design a nucleic acid probe toidentify and clone DNA encoding polypeptides having xylanase activityfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, preferably at least 25, more preferably at least 35, and mostpreferably at least 70 nucleotides in length. It is, however, preferredthat the nucleic acid probe is at least 100 nucleotides in length. Forexample, the nucleic acid probe may be at least 200 nucleotides,preferably at least 300 nucleotides, more preferably at least 400nucleotides, or most preferably at least 500 nucleotides in length. Evenlonger probes may be used, e.g., nucleic acid probes which are at least600 nucleotides, at least preferably at least 700 nucleotides, morepreferably at least 800 nucleotides, or most preferably at least 900nucleotides in length. Both DNA and RNA probes can be used. The probesare typically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having xylanaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, 3, or 5, or a subsequence thereof, thecarrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 1, 3, or 5,the cDNA sequence contained in SEQ ID NO: 1, 3, or 5, its complementarystrand, or a subsequence thereof, under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another preferred embodiment, the nucleic acid probe is SEQID NO: 1. In another preferred embodiment, the nucleic acid probe is themature polypeptide coding region of SEQ ID NO: 1. In another preferredembodiment, the nucleic acid probe is the nucleic acid sequencecontained in plasmid pSMO210 which is contained in Escherichia coli NRRLB-30706, wherein the nucleic acid sequence encodes a polypeptide havingxylanase activity. In another preferred embodiment, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpSMO210 which is contained in Escherichia coli NRRL B-30706.

In another preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO: 4, or asubsequence thereof. In another preferred embodiment, the nucleic acidprobe is SEQ ID NO: 3. In another preferred embodiment, the nucleic acidprobe is the mature polypeptide coding region of SEQ ID NO: 3. Inanother preferred embodiment, the nucleic acid probe is the nucleic acidsequence contained in plasmid pJLin162 which is contained in Escherichiacoli NRRL B-30702, wherein the nucleic acid sequence encodes apolypeptide having xylanase activity. In another preferred embodiment,the nucleic acid probe is the mature polypeptide coding region containedin plasmid pJLin162 which is contained in Escherichia coli NRRL B-30702.

In a preferred embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the polypeptide of SEQ ID NO: 6, or a subsequencethereof. In another preferred embodiment, the nucleic acid probe is SEQID NO: 5. In another preferred embodiment, the nucleic acid probe is themature polypeptide coding region of SEQ ID NO: 5. In another preferredembodiment, the nucleic acid probe is the nucleic acid sequencecontained in plasmid pHyGe009 which is contained in Escherichia coliNRRL B-30703, wherein the nucleic acid sequence encodes a polypeptidehaving xylanase activity. In another preferred embodiment, the nucleicacid probe is the mature polypeptide coding region contained in plasmidpHyGe009 which is contained in Escherichia coli NRRL B-30703.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of SEQ ID NO: 2, 4, or 6, or a homologoussequence thereof; or the mature polypeptide thereof. Preferably, aminoacid changes are of a minor nature, that is conservative amino acidsubstitutions or insertions that do not significantly affect the foldingand/or activity of the protein; small deletions, typically of one toabout 30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up toabout 20-25 residues; or a small extension that facilitates purificationby changing net charge or another function, such as a poly-histidinetract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,xylanase activity) to identify amino acid residues that are critical tothe activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides which are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of amino acids 18 to 364 of SEQ ID NO: 2, amino acids 20 to323 of SEQ ID NO: 4, or amino acids 20 to 397 of SEQ ID NO: 6 is 10,preferably 9, more preferably 8, more preferably 7, more preferably atmost 6, more preferably 5, more preferably 4, even more preferably 3,most preferably 2, and even most preferably 1.

Sources of Polypeptides Having Xylanase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

A polypeptide of the present invention may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having xylanaseactivity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride polypeptide.

In a more preferred embodiment, the polypeptide is an Aspergillusfumigatus polypeptide, e.g., the polypeptide with the amino acidsequence of SEQ ID NO: 2, 4, or 6.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encode a polypeptide of the present invention.

In a preferred aspect, the nucleic acid sequence is set forth in SEQ IDNO: 1. In another more preferred embodiment, the nucleic acid sequenceis the sequence contained in plasmid pSMO210 that is contained inEscherichia coli NRRL B-30706. In another preferred aspect, the nucleicacid sequence is the mature polypeptide coding region of SEQ ID NO: 1.In another more preferred aspect, the nucleic acid sequence is themature polypeptide coding region contained in plasmid pSMO210 that iscontained in Escherichia coli NRRL B-30706. The present invention alsoencompasses nucleic acid sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof,which differ from SEQ ID NO: 1 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO: 1 which encode fragments of SEQ ID NO: 2 that have xylanaseactivity.

In another preferred embodiment, the nucleic acid sequence is set forthin SEQ ID NO: 3. In another more preferred embodiment, the nucleic acidsequence is the sequence contained in plasmid pJLin162 that is containedin Escherichia coli NRRL B-30702. In another preferred embodiment, thenucleic acid sequence is the mature polypeptide coding region of SEQ IDNO: 3. In another more preferred embodiment, the nucleic acid sequenceis the mature polypeptide coding region contained in plasmid pJLin162that is contained in Escherichia coli NRRL B-30702. The presentinvention also encompasses nucleic acid sequences which encode apolypeptide having the amino acid sequence of SEQ ID NO: 4 or the maturepolypeptide thereof, which differ from SEQ ID NO: 3 by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 3 which encode fragments of SEQ ID NO: 4 thathave xylanase activity.

In a preferred embodiment, the nucleic acid sequence is set forth in SEQID NO: 5. In another more preferred embodiment, the nucleic acidsequence is the sequence contained in plasmid pHyGe009 that is containedin Escherichia coli NRRL B-30703. In another preferred embodiment, thenucleic acid sequence is the mature polypeptide coding region of SEQ IDNO: 5. In another more preferred embodiment, the nucleic acid sequenceis the mature polypeptide coding region contained in plasmid pHyGe009that is contained in Escherichia coli NRRL B-30703. The presentinvention also encompasses nucleic acid sequences which encode apolypeptide having the amino acid sequence of SEQ ID NO: 6 or the maturepolypeptide thereof, which differ from SEQ ID NO: 5 by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 5 which encode fragments of SEQ ID NO: 6 thathave xylanase activity.

The present invention also relates to mutant polunucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, 3, or 5, in which the mutant nucleotide sequence encodes apolypeptide which consists of amino acids 18 to 364 of SEQ ID NO: 2,amino acids 20 to 323 of SEQ ID NO: 4, or amino acids 20 to 397 of SEQID NO: 6, respectively.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Aspergillus, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 (i.e., nucleotides 52 to 1145 of SEQ IDNO: 1) of at least 65%, preferably at least 70%, more preferably atleast 75%, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, even more preferably at least 95%, andmost preferably at least 97%, 98%, or 99% identity, which encode anactive polypeptide.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 3 (i.e., nucleotides 58 to 1400) of atleast 85%, preferably at least 90%, more preferably at least 95%, andmost preferably at least 97%, 98%, or 99% identity, which encode anactive polypeptide.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 5 (i.e., nucleotides 107 to 1415) of atleast 80%, preferably at least 85%, more preferably at least 90%, evenmore preferably at least 95%, and most preferably at least 97%, 98%, or99% identity, which encode an active polypeptide.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 1, 3, or 5, e.g., a subsequence thereof, and/or by introductionof nucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forxylanase activity to identify amino acid residues that are critical tothe activity of the molecule. Sites of substrate-enzyme interaction canalso be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) nucleotides52 to 1145 of SEQ ID NO: 1, nucleotides 58 to 1400 of SEQ ID NO: 3, ornucleotides 107 to 1415 of SEQ ID NO: 5, (ii) the cDNA sequencecontained in nucleotides 52 to 1145 of SEQ ID NO: 1, nucleotides 58 to1400 of SEQ ID NO: 3, or nucleotides 107 to 1415 of SEQ ID NO: 5, or(iii) a complementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 52 to 1145 of SEQ ID NO: 1, nucleotides 58 to 1400 of SEQ IDNO: 3, or nucleotides 107 to 1415 of SEQ ID NO: 5, (ii) the cDNAsequence contained in nucleotides 52 to 1145 of SEQ ID NO: 1,nucleotides 58 to 1400 of SEQ ID NO: 3, or nucleotides 107 to 1415 ofSEQ ID NO: 5, or (iii) a complementary strand of (i) or (ii); and (b)isolating the hybridizing polynucleotides, which encode a polypeptidehaving xylanase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 51 of SEQ ID NO: 1 which encode amino acids 1 to 17 of SEQ ID NO: 2.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 57 of SEQ ID NO: 3 which encode amino acids 1 to 19 of SEQ ID NO: 4.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 106 of SEQ ID NO: 5, or the cDNA thereof, which encode amino acids 1to 19 of SEQ ID NO: 6.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phiebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating acell, which in its wild-type form is capable of producing thepolypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. Preferably, the cell isof the genus Aspergillus and more preferably Aspergillus fumigatus.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding region of SEQ ID NO:1, wherein the mutant nucleotide sequence encodes a polypeptide whichconsists of amino acids 18 to 364 of SEQ ID NO: 2, amino acids 20 to 323of SEQ ID NO: 4, or amino acids 20 to 397 of SEQ ID NO: 6, and (b)recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having xylanase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome or chloroplastgenome and propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having xylanase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Xylanase Activity

The present invention also relates to methods for producing a mutant ofa parent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or morenucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleotidesequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence which is then transformed into theparent cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred embodiment, the nucleotide sequence is disruptedwith a selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising: (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of xylanase activity byfermentation of a cell which produces both a polypeptide of the presentinvention as well as the protein product of interest by adding aneffective amount of an agent capable of inhibiting xylanase activity tothe fermentation broth before, during, or after the fermentation hasbeen completed, recovering the product of interest from the fermentationbroth, and optionally subjecting the recovered product to furtherpurification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of xylanase activity bycultivating the cell under conditions permitting the expression of theproduct, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the xylanase activitysubstantially, and recovering the product from the culture broth.Alternatively, the combined pH and temperature treatment may beperformed on an enzyme preparation recovered from the culture broth. Thecombined pH and temperature treatment may optionally be used incombination with a treatment with a xylanase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the xylanase activity. Complete removal of xylanase activity maybe obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH of 4-5 and a temperature of 80-90° C. for a sufficient period of timeto attain the desired effect, where typically, 30 to 60 minutes issufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyxylanase-free product is of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The enzyme may be selected from, e.g., an amylolytic enzyme, lipolyticenzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plantcell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, galactosidase, beta-galactosidase,glucoamylase, glucose oxidase, alpha- or beta-glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or another xylanase. Thexylanase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from xylanase activity which is produced by a method ofthe present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thexylanase activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, or nother xylanase. Theadditional enzyme(s) may be produced, for example, by a microorganismbelonging to the genus Aspergillus, preferably Aspergillus aculeatus,Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, orAspergillus oryzae; Fusarium, preferably Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium suiphureum, Fusariumtoruloseum, Fusarium trichothecioides, or Fusarium venenatum; Humicola,preferably Humicola insolens or Humicola lanuginosa; or Trichoderma,preferably Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

A polypeptide having xylanase activity of the present invention may beused in several applications to degrade or convert a xylan-containingmaterial by treating the material with an effective amount of thepolypeptide (see, for example, WO 2002/18561).

The polypeptides may be used in methods for the treatment of pulpaccording to U.S. Pat. No. 5,658,765.

The polypeptides may also be used in processes for producing xylose orxylo-oligosaccharide according to U.S. Pat. No. 5,658,765.

The polypeptides may also be used as feed enhancing enzymes that improvefeed digestibility to increase the efficiency of its utilizationaccording to U.S. Pat. No. 6,245,546.

The polypeptides may also be used in baking according to U.S. Pat. No.5,693,518.

The polypeptides may further be used in brewing according to WO2002/24926.

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to a nucleotide sequenceconsisting of a signal peptide consisting of nucleotides 1 to 51 of SEQID NO: 1, nucleotides 1 to 57 of SEQ ID NO: 3, or nucleotides 1 to 106of SEQ ID NO: 5, or the cDNA thereof, encoding a signal peptideconsisting of amino acids 1 to 17 of SEQ ID NO: 2, amino acids 1 to 19of SEQ ID NO: 4, or amino acids 1 to 19 of SEQ ID NO: 6, respectively,which allows secretion of the protein into a culture medium, wherein thegene is foreign to the nucleotide sequence.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising: (a) cultivating such a recombinant host cell underconditions suitable for production of the protein; and (b) recoveringthe protein.

The first and second nucleotide sequences may be operably linked toforeign genes individually with other control sequences or incombination with other control sequences. Such other control sequencesare described supra. As described earlier, where both signal peptide andpropeptide regions are present at the amino terminus of a protein, thepropeptide region is positioned next to the amino terminus of a proteinand the signal peptide region is positioned next to the amino terminusof the propeptide region.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

Examples Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Aspergillus oryzae BECh2 strain (Δalp, Δamy, CPA-, KA-, Δnp1) was usedfor expression of the Aspergillus fumigatus xylanase. Aspergillusfumigatus PaHa34 was used as the source of the Family 10 xylanase.

Media

Minimal medium was composed per liter of 6 g of NaNO₃, 0.52 g of KCl,1.52 g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g of Nobleagar, 1% glucose, and 0.5% MgSO₄.7H₂O.

COVE plates were composed per liter of 342.3 g of sucrose, 20 ml of COVEsalt solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl₂, and 25 g ofNoble agar.

COVE salt solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals solution.

COVE trace elements solution was composed per liter of 0.04 g ofNa₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

MY25 medium was composed per liter of 25 g of maltodextrin, 2 g ofMgSO₄.7H₂O, 10 g of KH₂PO₄, 2 g of citric acid, 2 g of K₂SO₄, 2 g ofurea, 10 g of yeast extract, and 1.5 ml of AMG trace metals solution,adjusted to pH 6.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, and 3 g of citric acid.

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of NaCl.

2× YT medium was composed per liter of 16 g of tryptone, 10 g of yeastextract, and 5 g of NaCl. 2× YT plates were composed per liter of 16 gof tryptone, 10 g of yeast extract, 5 g of NaCl and 15 g of Noble agar.

SOC medium was composed per liter of 20 g of tryptone, 5 g of yeastextract, 2 ml of 5 M NaCl, and 2.5 ml of 1 M KCl.

Example 1: Identification of a Family GH10 Xylanase Gene in the GenomicSequence of Aspergillus fumigatus

A tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods andProtocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) of theAspergillus fumigatus partial genome sequence (The Institute for GenomicResearch, Rockville, Md.) was carried out using as query a Family 10xylanase protein sequence from Aspergillus kawachii (Accession No.P33559). Several genes were identified as putative Family GH10 homologsbased upon a high degree of similarity to the query sequence at theamino acid level. Three genomic regions of approximately 3000 bp withgreater than 70% identity to the query sequence at the amino acid levelwere identified.

Example 2: Aspergillus fumigatus Genomic DNA Extraction

Aspergillus fumigatus was grown in 250 ml of potato dextrose medium in abaffled shake flask at 37° C. and 240 rpm. Mycelia were harvested byfiltration, washed twice in TE (10 mM Tris-1 mM EDTA), and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, which was resuspended in pH 8.0 buffer containing 10 mMTris, 100 mM EDTA, 1% TRITON® X-100, 0.5 M guanidine-HCl, and 200 mMNaCl. DNase-free RNase A was added at a concentration of 20 mg per literand the lysate was incubated at 37° C. for 30 minutes. Cellular debriswas removed by centrifugation, and DNA was isolated by using QIAGEN®Maxi 500 columns (QIAGEN Inc., Valencia, Calif.). The columns wereequilibrated in 10 ml of QBT washed with 30 ml of QC, and eluted with 15ml of QF (all buffers from QIAGEN Inc., Valencia, Calif.). DNA wasprecipitated in isopropanol, washed in 70% ethanol, and recovered bycentrifugation. The DNA was resuspended in TE buffer.

Example 3: Construction of pAlLo1 Expression Vector

Expression vector pAlLo1 was constructed by modifying pBANe6 (U.S. Pat.No. 6,461,837), which comprises a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase (NA2-tpi promoter), Aspergillus nigeramyloglucosidase terminator sequence (AMG terminator), and Aspergillusnidulans acetamidase gene (amdS). All mutagenesis steps were verified bysequencing using BIGDYE™ terminator chemistry (Applied Biosystems, Inc.,Foster City, Calif.). Modification of pBANe6 was performed by firsteliminating three Nco I restriction sites at positions 2051, 2722, and3397 bp from the amdS selection marker by site-directed mutagenesis. Allchanges were designed to be “silent” leaving the actual protein sequenceof the amdS gene product unchanged. Removal of these three sites wasperformed simultaneously with a GENEEDITOR™ in vitro Site-DirectedMutagenesis Kit (Promega, Madison, Wis.) according to the manufacturer'sinstructions using the following primers (underlined nucleotiderepresents the changed base):

AMDS3NcoMut (2050): (SEQ ID NO: 7) 5′-GTGCCCCATGATACGCCTCCGG-3′AMDS2NcoMut (2721): (SEQ ID NO: 8) 5′-GAGTCGTATTTCCAAGGCTCCTGACC-3′AMDS1NcoMut (3396): (SEQ ID NO: 9) 5′-GGAGGCCATGAAGTGGACCAACGG-3′

A plasmid comprising all three expected sequence changes was thensubmitted to site-directed mutagenesis, using a QUICKCHANGE™Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.), toeliminate the Nco I restriction site at the end of the AMG terminator atposition 1643. The following primers (underlined nucleotide representsthe changed base) were used for mutagenesis:

Upper Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 10)5′-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGAC AG-3′Lower Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 11)5′-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTC TG-3′

The last step in the modification of pBANe6 was the addition of a newNco I restriction site at the beginning of the polylinker using aQUICKCHANGE™ Site-Directed Mutagenesis Kit and the following primers(underlined nucleotides represent the changed bases) to yield pAlLo1(FIG. 2).

Upper Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 12)5′-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3′Lower Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 13)5′-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3′

Example 4: Construction of pBM120a Expression Vector

Plasmid pBM120a was constructed to obtain a plasmid containing a doubleNA2 promoter (NA2-NA2-tpi) for driving gene expression in Aspergillusspecies, and containing the ampicillin resistance gene for selection inE. coli.

Primers were designed to PCR amplify the double NA2 promoter frompJaL721 (WO 03/008575). Restriction enzyme sites Sal I and Nco I(underlined) were added for cloning the double promoter into theAspergillus expression plasmid pAlLo1.

(SEQ ID NO: 14) 5′-GTCGACATGGTGTTTTGATCATTTTA-3′ (SEQ ID NO: 15)5′-CCATGGCCAGTTGTGTATATAGAGGA-3′

The fragment of interest was amplified by PCR using the EXPAND® HighFidelity PCR System (Roche Diagnostics, Mannheim, Germany). The PCRamplification reaction mixture contained 1 μl of 0.09 μg of pJaL721, 1μl of each of the primers (50 pmol/μl), 5 μl of 10×PCR buffer with 15 mMMgCl₂, 1 μl of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), 37.25 μlof water, and 0.75 μl (3.5 U/μl) of DNA polymerase mix. An EPPENDORF®MASTERCYCLER® thermocycler was used to amplify the fragment with thefollowing settings: 1 cycle at 94° C. for 2 minutes; 10 cycles each at94° C. for 15 seconds, 55° C. for 30 seconds, 72° C. for 1.25 minutes;15 cycles each at 94° C. for 15 seconds, 55° C. for 30 seconds, 72° C.for 1.25 minutes plus 5 second elongation at each successive cycle; 1cycle at 72° C. for 7 minutes; and a 10° C. hold. Ten microliters ofthis PCR reaction was mixed with 1 μl of 10×DNA loading dye (25%glycerol, 10 mM Tris pH 7.0, 10 mM EDTA, 0.025% bromophenol blue, 0.025%xylene cyanol) and run on a 1.0% (w/v) agarose gel using 40 mM Trisbase-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer. An 1128 bpPCR product was observed with UV light on a NUCLEOTECH® gelvisualization system (Nucleotech, San Mateo, Calif.). The PCR productwas directly ligated into pCR®2.1-TOPO (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions. A 1 μl volume of fresh PCRproduct, 3 μl of double-distilled water, and 1 μl of the TOPO cloningvector were mixed with a pipette and incubated on the bench top for 5minutes.

After the incubation, 2 μl of the mixture was used to transform ONESHOT® competent E. coli cells (Invitrogen, Carlsbad, Calif.). A 2 μlvolume of the ligation mixture was added to the E. coli cells andincubated on ice for 5 minutes. Subsequently, the cells were heatshocked for 30 seconds at 42° C., and then placed on ice for 2 minutes.A 250 μl volume of SOC medium was added to these cells and the mixturewas incubated for 1 hour at 37° C. and 250 rpm. After the incubation thecolonies were spread on 2× YT plates supplemented with 100 μg ofampicillin per ml and incubated at 37° C. overnight for selection of theplasmid. Eight colonies that grew on the plates were picked with asterile toothpick and grown overnight at 37° C., 250 rpm in a 15 mlFALCON® tube containing 3 ml of LB medium supplemented with 100 μg ofampicillin per ml. The plasmids were isolated using the QIAGEN® robotprotocol (QIAGEN, Valencia, Calif.).

Four μl volumes of the resulting plasmid minipreps were digested withEco RI. The digestion reactions were analyzed by agarose gelchromatography and UV analysis as previously described for the PCRreaction. Isolated plasmids containing an insert were sequenced using 1μl of plasmid template, 1.6 ng of M13 primer (forward or reverse) (MWGBiotech; High Point; NC), and water to 6 μl. DNA sequencing wasperformed with an APPLIED BIOSYSTEMS® Model 377 Sequencer XL usingdye-terminator chemistry. The resulting plasmid was designated pBM121b(FIG. 3).

A 5 μl volume of pBM121b was digested with Sal I and Nco I. Thedigestion reactions were analyzed by agarose gel electrophoresis asdescribed above, and ligated to the vector pAlLo1, which had beenpreviously cleaved with Sal I and Nco I. The resulting expressionplasmid was designated pBM120a (FIG. 4).

Example 5: Cloning of a Family GH10A Xylanase Gene and Construction ofan Aspergillus oryzae Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify an Aspergillus fumigatus gene encoding a Family GH10A xylanasegene from the genomic DNA prepared in Example 2. An IN-FUSION® CloningKit (BD Biosciences, Palo Alto, Calif.) was used to clone the fragmentdirectly into the expression vector pBM120a without the need forrestriction digests and ligation.

Forward primer: (SEQ ID NO: 16) 5′-TACACAACTGGCCATGCGTTTCTCCCTTGCCGC-3′Reverse primer: (SEQ ID NO: 17)5′-AGTCACCTCTAGTTAATTAACTAGCATACAGTGCAGGGCT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to insertion sites of pBM120a.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 100 ng of Aspergillus fumigatus genomic DNA, 1× EXPAND® HighFidelity Amplification Buffer (Roche, Indianapolis, Ind.), 1.5 μl of a10 mM blend of dATP, dTTP, dGTP, and dCTP, and 2.5 units of EXPAND® HighFidelity Polymerase (Roche, Indianapolis, Ind.), in a final volume of 50μl. An EPPENDORF® MASTERCYCLER® thermocycler was used to amplify thefragment with the following settings: one cycle at 94° C. for 2 minutes;and 30 cycles each at 94° C. for 15 seconds, 60° C. for 30 seconds, and70° C. for 3 minutes. The heat block then went to a 10° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 1.1 kb product band was excised from the gel and purifiedusing a QIAQUICK® Gel Extraction Kit (QIAGEN, Valencia, Calif.)according to the manufacturer's instructions.

The fragment was then cloned into the expression vector pBM120a using anIN-FUSION® Cloning Kit. The vector was digested with Nco I and Pac I.The fragment was purified by agarose gel electrophoresis and QIAQUICK®gel purification as previously described. The gene fragment and digestedvector were ligated together in a reaction resulting in the expressionplasmid pSMO₂₀₈ (FIG. 5) in which transcription of the Family GH10Axylanase gene was under the control of the NA2-NA2-tpi promoter. Theligation reaction (20 μl) was composed of 1× IN-FUSION® Buffer (BDBiosciences, Palo Alto, Calif.), 1×BSA (BD Biosciences, Palo Alto,Calif.), 1 μl of IN-FUSION® enzyme (diluted 1:10) (BD Biosciences, PaloAlto, Calif.), 100 ng of pBM120a digested with Nco I and Pac I, and 100ng of the Aspergillus fumigatus xylanase purified PCR product. Thereaction was incubated at room temperature for 30 minutes. One μl of thereaction was used to transform E. coli XL10 SOLOPACK® Gold cells(Stratagene, La Jolla, Calif.). An E. coli transformant containingpSMO₂₀₈ was detected by restriction digestion and plasmid DNA wasprepared using a BIOROBOT® 9600 (QIAGEN, Inc., Valencia, Calif.)according to the manufacturer's instructions.

Example 6: Characterization of the Aspergillus fumigatus GenomicSequence Encoding a Family GH10A Xylanase

DNA sequencing of the Family GH10A Aspergillus fumigatus xylanase genefrom pSMO₂₀₈ was performed with an APPLIED BIOSYSTEMS® Model 377 XLAutomated DNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., FosterCity, Calif.) using dye-terminator chemistry (Giesecke et al., 1992,Journal of Virology Methods 38: 47-60) and primer walking strategy.Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash.).

Sequence analysis of pSMO₂₀₈ revealed 24 base-pair changes from thepredicted sequence. Translation of the DNA sequence to amino acidsresulted in 8 amino acid changes, including a stop codon at amino acidposition 130. Site-directed mutagenesis was used to remove the stopcodon and change one other amino acid to be more consistent withxylanase consensus sequences.

Example 7: Site-Directed Mutagenesis of the Family GH10A Xylanase Geneand Construction of an Aspergillus oryzae Expression Vector

To eliminate the stop codon at amino acid position 130, two syntheticoligonucleotide primers shown below were designed to PCR amplify theAspergillus fumigatus GH10A xylanase gene containing a single bp changeusing a QUIKCHANGE® IIXL Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.).

(SEQ ID NO: 18) 5′-CCTCCAGGAACTGGACCGCCACAGAACTC-3′ (SEQ ID NO: 19)5′-GAGTTCTGTGGCGGTCCAGTTCCTGGAGG-3′

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 10 ng of pSMO₂₀₈, 1× QUIKCHANGE® Amplification buffer(Stratagene, La Jolla, Calif.), 3 μl of QUIKCHANGE® Solution reagent, 1μl of a 10 mM blend of dATP, dTTP, dGTP, and dCTP, and 2.5 units ofEXPAND® High Fidelity Polymerase, in a final volume of 50 μl. AnEPPENDORF® MASTERCYCLER® thermocycler was used to amplify the fragmentwith the following settings: one cycle at 95° C. for 2 minutes; 18cycles each at 95° C. for 50 seconds, 60° C. for 50 seconds, and 68° C.for 8 minutes; and 1 cycle at 68° C. for 7 minutes. The heat block thenwent to a 10° C. soak cycle. Dpn I was added directly to theamplification reaction and incubated at 35° C. for 1 hour. A 2 μl volumeof the Dpn I digested reaction was used to transform E. coli XL10 GOLD®Ultra competent cells. Plasmid DNA was prepared from an E. colitransformant using a BIOROBOT® 9600.

Sequence analysis verified the single base pair change removing the stopcodon, resulting in pSMO₂₀₉. To change the amino acid at amino acidposition 169, two oligonucleotides were constructed as shown below.

997144: (SEQ ID NO: 20) 5′-GCTATTAATGGGGACGGGACCTTTTCCTCCAGTGTG-3′997145: (SEQ ID NO: 21) 5′-CACACTGGAGGAAAAGGTCCCGTCCCCATTAATAGC-3′

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 10 ng of pSMO₂₀₉, 1× QUIKCHANGE® Amplification buffer, 3 μlof QUIKCHANGE® Solution reagent, 1 μl of a 10 mM blend of dATP, dTTP,dGTP, and dCTP, and 2.5 units of EXPAND® High Fidelity DNA Polymerase,in a final volume of 50 μl. An EPPENDORF® MASTERCYCLER® thermocycler wasused to amplify the fragment with the following settings: one cycle at95° C. for 2 minutes; 18 cycles each at 95° C. for 50 seconds, 60° C.for 50 seconds, and 68° C. for 8 minutes; and 1 cycle at 68° C. for 7minutes. The heat block then went to a 10° C. soak cycle. Dpn I wasdirectly added to the amplification reaction and incubated at 35° C. for1 hour. A 2 μl volume of the Dpn I digested reaction was used totransform E. coli XL10 GOLD® Ultracompetent cells. Sequence analysisverified the base change resulting in pSMO₂₁₀ (FIG. 6).

E. coli SoloPack Gold cells (Stratagene, La Jolla, Calif.) containingplasmid pSMO₂₁₀ was deposited with the Agricultural Research ServicePatent Culture Collection, Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, as NRRL B-30706, with a depositdate of Feb. 6, 2004.

Example 8: Characterization of the Aspergillus fumigatus GenomicSequence Encoding a Family GH10A Xylanase

DNA sequencing of the Aspergillus fumigatus GH10A xylanase gene frompSMO₂₁₀ was performed with an APPLIED BIOSYSTEMS® Model 377 XL AutomatedDNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City,Calif.) using dye-terminator chemistry (Giesecke et al., 1992, supra)and primer walking strategy. Nucleotide sequence data were scrutinizedfor quality and all sequences were compared to each other withassistance of PHRED/PHRAP software.

A gene model for the Aspergillus fumigatus GH10A sequence of pSMO₂₁₀ wasconstructed based on similarity to a homologous xylanase gene fromMyceliophthora thermophila (accession number NP000134). The nucleotidesequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2)are shown in FIGS. 1A and 1B. The genomic fragment encodes a polypeptideof 364 amino acids, interrupted by one 50 bp intron. The % G+C contentof the gene is 55.8%. Using the SignalP software program (Nielsen etal., 1997, Protein Engineering 10: 1-6), a signal peptide of 17 residueswas predicted. The predicted mature protein contains 347 amino acidswith a molecular mass of 40.4 kDa.

A comparative alignment of xylanase sequences was determined using theClustal W method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE®MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity tableand the following multiple alignment parameters: Gap penalty of 10 andgap length penalty of 10. Pairwise alignment parameters were Ktuple=1,gap penalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Family GH10A Aspergillus fumigatusxylanase shares 36% identity to the deduced amino acid sequence of theMyceliophthora thermophila xylanase (Accession Number NP000134).

Example 9: Cloning of a Family GH10B Xylanase Gene and Construction ofan Aspergillus oryzae Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a Aspergillus fumigatus gene encoding a Family GH10B xylanasegene from the genomic DNA prepared in Example 2. An IN-FUSION® CloningKit was used to clone the fragment directly into the expression vector,pBM120a, without the need for restriction digests and ligation.

Forward primer: (SEQ ID NO: 22)5′-ACACAACTGGCCATGGTCGTCCTCAGCAAGCTCGTCA-3′ Reverse primer:(SEQ ID NO: 23) 5′-AGTCACCTCTAGTTAATTAATCAGAGAGCAGCAATGATGG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

The fragment of interest was amplified by PCR using the EXPAND® HighFidelity PCR System according to manufacturer's instructions. Each PCRreaction contained 250 ng of the genomic DNA template, 200 μM dATP,dTTP, dGTP, and dCTP mix, 1 μM forward and reverse primers, 1× reactionbuffer, and 2.6 units of EXPAND® High Fidelity enzyme mix in a finalvolume of 50 μl. An EPPENDORF® MASTERCYCLER® thermocycler was used toamplify the fragment with the following settings: 1 cycle at 94° C. for2 minutes; 10 cycles each at 94° C. for 15 seconds, 60° C. for 30seconds, and 72° C. for 1.25 minutes; 15 cycles each at 94° C. for 15seconds, 60° C. for 30 seconds, and 72° C. for 1.25 minutes plus 5second elongation at each successive cycle; 1 cycle at 72° C. for 7minutes; and a 10° C. hold.

The reaction products were isolated on a 0.7% agarose gel using 50 mMTris base-50 mM boric acid-1 mM disodium EDTA (TBE) buffer and a 1.4 kbproduct band was excised from the gel and purified using a QIAQUICK® GelExtraction Kit according to the manufacturer's instructions.

The fragment was then cloned into the expression vector pBM120a using anIN-FUSION® Cloning Kit. The vector was digested with Nco I and Pac I.Both the digested vector and PCR fragment were purified by gelelectrophoresis and QIAQUICK® gel extraction as previously described.The gene fragment and digested vector were ligated together in areaction resulting in the expression plasmid pJLin162 (FIG. 8) in whichtranscription of the Family GH10B xylanase gene was under the control ofthe NA2-NA2-tpi promoter. The ligation reaction (50 μl) was composed of1× IN-FUSION® Buffer, 1×BSA, 1 μl of IN-FUSION® enzyme (diluted 1:10),100 ng of pBM120a digested with Nco I and Pac I, and 50 ng of theAspergillus fumigatus xylanase purified PCR product. The reaction wasincubated at room temperature for 30 minutes. Two μl of the reaction wasused to transform E. coli SOLOPACK® Gold supercompetent cells(Stratagene, La Jolla, Calif.). An E. coli transformant containing thepJLin162 μlasmid was detected by restriction digestion and plasmid DNAwas prepared using a BIOROBOT® 9600.

E. coli SOLOPACK® Gold cells containing plasmid pJLin162 were depositedwith the Agricultural Research Service Patent Culture Collection,Northern Regional Research Center, 1815 University Street, Peoria, Ill.,61604, as NRRL B-30702, with a deposit date of Jan. 27, 2004.

Example 10: Characterization of the Aspergillus fumigatus GenomicSequence Encoding a Family GH10B Xylanase

DNA sequencing of the Aspergillus fumigatus GH10B xylanase gene frompJLin162 was performed with an APPLIED BIOSYSTEMS® Model 377 XLAutomated DNA Sequencer using dye-terminator chemistry (Giesecke et al.,1992, supra) and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software.

A gene model for the sequence was constructed based on the tfasty outputand alignment with an Aspergillus oryzae xynF1 (Accession numberAB011212). A comparative alignment of amino acid sequences wasdetermined using the MAFFT method with iterative refinement and defaultparameters (Katoh et al., 2002, Nucleic Acids Research 30: 3059). Thenucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQID NO: 4) are shown in FIGS. 7A and 7B. The genomic fragment encodes apolypeptide of 323 amino acids, interrupted by 8 introns of 57, 51, 56,52, 55, 58, 49 and 52 bp. The % G+C content of the gene is 55.5%. Usingthe SignalP software program (Nielsen et al., 1997, supra), a signalpeptide of 19 residues was predicted. The predicted mature proteincontains 304 amino acids with a molecular mass of 33 kDa.

A comparative alignment of xylanase sequences was determined using theClustal W method (Higgins, 1989, supra) using the LASERGENE® MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus fumigatus GH10B xylanasegene shares 74% identity to the deduced amino acid sequence of theAspergillus oryzae xylanase xynF1 (accession number AB011212).

Example 11: Expression of the Aspergillus fumigatus Family GH10BXylanase Gene in Aspergillus oryzae BECh2

Aspergillus oryzae BECh2 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Two μgof pJLin162, which was digested with Pme I to remove the ampicillinresistance gene, was used to transform Aspergillus oryzae BECh2.

The transformation of Aspergillus oryzae BECh2 with pJLin162 yieldedabout 300 transformants. Forty-two transformants were transferred toindividual COVE plates. Plugs of the Aspergillus oryzae BECh2transformants along with the parental strain (as a negative control)were transferred to Minimal medium plates (adjusted to pH 5) containing0.1% AZCL-arabinoxylan substrate (Megazyme, Ireland), and incubated at37° C. overnight. All 42 transformants formed blue zones around theplugs, indicating expression of xylanase activity. Spores of the 42transformants were then streaked to new COVE plates, followed by pickingsingle colonies the next day. Two single colonies were picked for eachtransformant. The spore-purified clones were tested on AZCL-arabinoxylanplates again, and one xylanase-positive clone per each transformant wasspore-purified the second time as described above.

Spores of 34 of the 42 transformants were collected in 4 ml of 0.01%TWEEN® 20 and 200 μl of the spore suspension were inoculated separatelyinto 25 ml of MY25 medium in 125 ml plastic shake flasks and incubatedat 34° C., 250 rpm. Three, four, and five days after inoculation,culture supernatants were removed and assayed for xylanase activity.

Culture supernatants prepared as described above were subjected to assayfor xylanase activity as described below. Briefly, 135 μl of assaybuffer (400 mM sodium phosphate pH 6 buffer) were mixed with 135 μl of0.4% AZCL-arabinoxylan in 0.02% TRITON® X-100 for each reaction (with afinal concentration of 0.2% substrate in 0.01% TRITON® X-100 and 200 mMsodium phosphate buffer). Then 30 μl of the diluted supernatant sampleswere added. Dilutions of SHEARZYME® 500 L, a purified xylanase fromAspergillus aculeatus (obtained from Novozymes A/S, Bagsværd, Denmark),at 600 FXU (fungal xylanase unit) per ml were used as a standard. Afterhigh-speed (1400 rpm) mixing at 37° C. for 15 minutes in an EPPENDORF®Thermomixer (Brinkmann Instruments, NY), samples were placed on ice for2 minutes before centrifugation for 2 minutes at 1510×g. Aftercentrifugation, 150 μl samples of the supernatants were measured at 650nm. Xylanase activities of the transformants were determined by plottingthe A₆₅₀ value against the standard curve generated by SHEARZYME® 500 L.All of the transformants were found to express xylanase activity.SDS-PAGE (BIO-RAD® CRITERION® 10-20% SDS-PAGE) analysis of 0.5 μl of thesupernatants showed a major band at approximately 30 kDa.

Example 12: Cloning of a Family GH10C Xylanase Gene and Construction ofan Aspergillus oryzae Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a Aspergillus fumigatus gene encoding a Family GH10C xylanasegene from the genomic DNA prepared in Example 2. Vector pCR®2.1-TOPO(Invitrogen, Carlsbad, Calif.) was used to amplify the gene PCR product.The fragment of the gene was released by digesting with Nco I and Pac Iand then ligated to expression vector pBM120a using a Rapid DNA LigationKit (Boehringer Mannheim, Germany) resulting in the expression plasmidpHyGe001.

Forward primer: (SEQ ID NO: 24) 5′-CCATGGTCCATCTATCTTCATT-3′Reverse primer: (SEQ ID NO: 25) 5′-TTAATTAATTACAGGCACTGTGAGTACC-3′Bold letters represent coding sequence. The remaining sequence is addedfor cloning sites.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 100 ng of Aspergillus fumigatus genomic DNA, 1× EXPAND® HighFidelity Amplification Buffer, 1.5 μl of a 10 mM blend of dATP, dTTP,dGTP, and dCTP, and 2.5 units of EXPAND® High Fidelity Polymerase, in afinal volume of 50 μl. An EPPENDORF® MASTERCYCLER® thermocycler was usedto amplify the fragment with the following settings: one cycle at 94° C.for 2 minutes; 10 cycles each at 94° C. for 15 seconds, 56.8° C. for 30seconds, and 72° C. for 1 minute and 15 seconds; 15 cycles each at 94°C. for 15 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute and 15seconds plus 5 second elongation at each successive cycle; and 1 cycleat 72° C. for 7 minutes. The heat block then went to a 10° C. soakcycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 1.4 kb product band was excised from the gel and purifiedusing a QIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

The fragment was then cloned into the pCR2.1-TOPO vector. The genefragment was purified by a PCR Clean Up Kit (QIAGEN, Valencia, Calif.).The fragment and pCR®2.1-TOPO vector were ligated by using conditionsspecified by the manufacturer resulting in plasmid pHyGe009 (FIG. 10).Two μl of the reaction was used to transform E. coli ONE SHOT® competentcells (Invitrogen, Carlsbad, Calif.). An E. coli transformant containingthe plasmid pHyGe009 was detected by restriction digestion and plasmidDNA was prepared using a BIOROBOT® 9600.

The gene fragment from pHyGe009 was cloned into the pBM120a expressionvector. The gene fragment was released from pHyGe009 by digestion withNco I and Pac I and then purified by gel electrophoresis and QIAQUICK®gel purification as previously described. The pBM120a vector wasdigested with Nco I and Pac I. The gene fragment and the digested vectorwere ligated together using a Rapid DNA Ligation Kit resulting inexpression plasmid pHyGe001 (FIG. 11) in which transcription of theFamily GH10C xylanase gene was under the control of the NA2-NA2-tpipromoter. Five μl of the reaction was used to transform E. coli XL1-BlueSubcloning-Grade Competent Cells (Stratagene, La Jolla, Calif.). An E.coli transformant containing the pHyGe001 μlasmid was detected byrestriction digestion and plasmid DNA was prepared using a BIOROBOT®9600.

E. coli TOP10 containing plasmid pHyGe001 was deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30703, with a deposit date of Jan. 28, 2004.

Example 13: Characterization of the Aspergillus fumigatus GenomicSequence Encoding a Family GH10C Xylanase

DNA sequencing of the Aspergillus fumigatus GH10C xylanase gene frompHyGe009 was performed with an APPLIED BIOSYSTEMS® Model 3700 AutomatedDNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992,supra) and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software.

A gene model for the sequence was constructed based on the tfasty outputand alignment with an Aspergillus acu/eatus xylanase (Accession NumberP48825). A comparative alignment of amino acid sequences was determinedusing the MAFFT method with iterative refinement and default parameters(Katoh et al., 2002, supra). The nucleotide sequence (SEQ ID NO: 5) anddeduced amino acid sequence (SEQ ID NO: 6) are shown in FIGS. 9A and 9B.The genomic fragment encodes a polypeptide of 397 amino acids,interrupted by 4 introns of 49, 65, 48 and 59 bp. The % G+C content ofthe gene is 53%. Using the SignalP software program (Nielsen et al.,1997, supra), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 378 amino acids with a molecular massof 40 kDa. A cellulose binding domain was identified using InterProScansoftware (Zdobnov, E. M. and Apweiler, R., 2001, InterProScan—anintegration platform for the signature-recognition methods in InterPro,Bioinformatics 17(9): p. 847-8), which comprises 108 bp from nucleotide1305 to nucleotide 1412 encoding 36 amino acids with the deduced aminoacid sequence of VAQKWGQCGGIGWTGPTTCVSGTTCQKLNDWYSQCL (amino acids 362to 397 of SEQ ID NO: 6).

A comparative alignment of xylanase sequences was determined using theClustal W method (Higgins, 1989, supra) using the LASERGENE® MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus fumigatus GH10C xylanasegene shares 67.8% identity to the deduced amino acid sequence of theAspergillus aculeatus xylanase (Accession Number P48825).

Example 14: Expression of the Aspergillus fumigatus Family GH10CXylanase Gene in Aspergillus oryzae BECh2

Aspergillus oryzae BECh2 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. A 3.9 μg quantity ofpHyGe001, which was digested with PmeI to remove the ampicillinresistance gene, was used to transform Aspergillus oryzae BECh2.

The transformation of Aspergillus oryzae BECh2 with pHyGe001 yieldedabout 49 transformants. The 49 transformants were transferred toindividual COVE plates. Plugs of the Aspergillus oryzae BECh2transformants along with the parental strain (as a negative control)were transferred to Minimal medium plates (adjusted to pH 6) containing0.1% AZCL-arabinoxylan substrate (Megazyme, Ireland), and incubated at37° C. overnight. Forty-one transformants formed blue zones around theplugs, indicating expression of xylanase activity. Spores of the 41transformants were then streaked to new COVE plates, followed by pickingsingle colonies the next day. Two single colonies were picked for eachtransformant. The spore-purified clones were tested on AZCL-arabinoxylanplates again, and one xylanase-positive clone per each transformant wasspore-purified a second time as described above.

Spore stocks of the 41 of transformants were collected in 5 ml of 0.01%TWEEN® 20, and 200 μl of the spore suspension were inoculated into 25 mlof MY25 medium in 125 ml plastic shake flasks and incubated at 34° C.,250 rpm. Three, four, and five days after incubation, culturesupernatants were removed and assayed for xylanase activity.

Culture supernatants prepared as described above were subjected to thexylanase assay as described in Example 11. The assay resultsdemonstrated that all of the transformants expressed xylanase activity.SDS-PAGE (BIO-RAD® CRITERION® 10-20% SDS-PAGE) analysis of 10 μl of thesupernatants showed a major band at approximately 50 kDa.

Deposit of Biological Materials

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli (pSMO210) NRRL B-30706Feb. 6, 2004 E. coli (pJLin162) NRRL B-30702 Jan. 27, 2004 E. coli TOP10(pHyGe009) NRRL B-30703 Jan. 28, 2004

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent a substantially pure culture of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-60. (canceled)
 61. A nucleic acid construct comprising an isolatedpolynucleotide encoding a polypeptide having xylanase activity operablylinked to one or more heterologous control sequences that directproduction of the polypeptide in a recombinant expression host, whereinthe polynucleotide is selected from the group consisting of: (a) apolynucleotide encoding a polypeptide having xylanase activitycomprising an amino acid sequence having at least 95% identity to aminoacids 20 to 397 of SEQ ID NO: 6; (b) a polynucleotide encoding apolypeptide having xylanase activity which hybridizes under highstringency conditions with (i) nucleotides 107 to 1415 of SEQ ID NO: 5,(ii) the cDNA sequence of nucleotides 107 to 1415 of SEQ ID NO: 5, or(iii) the full-length complement of (i) or (ii), wherein th highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 50% formamide, and washing three times each for 15minutes in 2×SSC, 0.2% SDS at 65° C.; (c) a polynucleotide encoding apolypeptide having xylanase activity comprising a polynucleotidesequence having at least 95% identity to nucleotides 107 to 1415 of SEQID NO: 5; (d) a polynucleotide encoding a polypeptide having xylanaseactivity comprising amino acids 20 to 397 of SEQ ID NO: 6; and (e) apolynucleotide encoding a polypeptide having xylanase activitycomprising nucleotides 107 to 1415 of SEQ ID NO:
 5. 62. The nucleic acidconstruct of claim 61, wherein the polynucleotide encodes a polypeptidehaving xylanase activity comprising an amino acid sequence having atleast 95% identity to amino acids 20 to 397 of SEQ ID NO:
 6. 63. Thenucleic acid construct of claim 61, wherein the polynucleotide encodes apolypeptide having xylanase activity comprising an amino acid sequencehaving at least 97% identity to amino acids 20 to 397 of SEQ ID NO: 6.64. The nucleic acid construct of claim 61, wherein the polynucleotideencodes a polypeptide having xylanase activity comprising an amino acidsequence having at least 98% identity to amino acids 20 to 397 of SEQ IDNO:
 6. 65. The nucleic acid construct of claim 61, wherein thepolynucleotide encodes a polypeptide having xylanase activity comprisingan amino acid sequence having at least 99% identity to amino acids 20 to397 of SEQ ID NO:
 6. 66. The nucleic acid construct of claim 61, whereinthe polynucleotide encodes a polypeptide having xylanase activitycomprising the amino acid sequence of SEQ ID NO: 6, or a fragmentthereof having xylanase activity.
 67. The nucleic acid construct ofclaim 61, wherein the polynucleotide encodes a polypeptide havingxylanase activity comprising the amino acid sequence of SEQ ID NO: 6.68. The nucleic acid construct of claim 61, wherein the polynucleotideencodes a polypeptide having xylanase activity comprising amino acids 20to 397 of SEQ ID NO:
 6. 69. The nucleic acid construct of claim 61,wherein the polynucleotide comprises SEQ ID NO: 5 or nucleotides 107 to1415 of SEQ ID NO:
 5. 70. The nucleic acid construct of claim 61,wherein the polynucleotide comprises the polynucleotide sequencecontained in plasmid pHyGe001 which is contained in E. coli NRRLB-30703.
 71. A recombinant expression vector comprising the nucleic acidconstruct of claim
 61. 72. An isolated recombinant host cell comprisingthe nucleic acid construct of claim
 61. 73. A method for producing apolypeptide having xylanase activity comprising: (a) cultivating therecombinant host cell of claim 72 under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 74. Atransgenic plant, plant part or plant cell, which has been transformedwith the nucleic acid construct of claim
 61. 75. A method for producinga polypeptide having xylanase activity, comprising: (a) cultivating thetransgenic plant or the plant cell of claim 74 under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.
 76. An isolated recombinant host cell transformed with anucleic acid construct comprising an isolated polynucleotide encoding apolypeptide having xylanase activity operably linked to one or morecontrol sequences that direct production of the polypeptide in therecombinant host cell, wherein the polypeptide having xylanase activityis heterologous to the recombinant host cell and wherein thepolynucleotide is selected from the group consisting of: (a) apolynucleotide encoding a polypeptide having xylanase activitycomprising an amino acid sequence having at least 95% identity to aminoacids 20 to 397 of SEQ ID NO: 6; (b) a polynucleotide encoding apolypeptide having xylanase activity which hybridizes under highstringency conditions with (i) nucleotides 107 to 1415 of SEQ ID NO: 5,(ii) the cDNA sequence of nucleotides 107 to 1415 of SEQ ID NO: 5, or(iii) the full-length complement of (i) or (ii), wherein the highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 50% formamide, and washing three times each for 15minutes in 2×SSC, 0.2% SDS at 65° C.; (c) a polynucleotide encoding apolypeptide having xylanase activity comprising a polynucleotidesequence having at least 95% identity to nucleotides 107 to 1415 of SEQID NO: 5; (d) a polynucleotide encoding a polypeptide having xylanaseactivity comprising amino acids 20 to 397 of SEQ ID NO: 6; and (e) apolynucleotide encoding a polypeptide having xylanase activitycomprising nucleotides 107 to 1415 of SEQ ID NO:
 5. 77. The recombinanthost cell of claim 76, wherein the polynucleotide encodes a polypeptidehaving xylanase activity comprising an amino acid sequence having atleast 95% identity to amino acids 20 to 397 of SEQ ID NO:
 6. 78. Therecombinant host cell of claim 76, wherein the polynucleotide encodes apolypeptide having xylanase activity comprising an amino acid sequencehaving at least 97% identity to amino acids 20 to 397 of SEQ ID NO: 6.79. The recombinant host cell of claim 76, wherein the polynucleotideencodes a polypeptide having xylanase activity comprising an amino acidsequence having at least 98% identity to amino acids 20 to 397 of SEQ IDNO:
 6. 80. The recombinant host cell of claim 76, wherein thepolynucleotide encodes a polypeptide having xylanase activity comprisingan amino acid sequence having at least 99% identity to amino acids 20 to397 of SEQ ID NO:
 6. 81. The recombinant host cell of claim 76, whereinthe polynucleotide encodes a polypeptide having xylanase activitycomprising the amino acid sequence of SEQ ID NO: 6, or a fragmentthereof having xylanase activity.
 82. The recombinant host cell of claim76, wherein the polynucleotide encodes a polypeptide having xylanaseactivity comprising the amino acid sequence of SEQ ID NO:
 6. 83. Therecombinant host cell of claim 76, wherein the polynucleotide encodes apolypeptide having xylanase activity comprising amino acids 20 to 397 ofSEQ ID NO:
 6. 84. A method for producing a polypeptide having xylanaseactivity comprising: (a) cultivating the recombinant host cell of claim76 under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.